Pro-apoptotic Cleavage Products of Bcl-xL Form Cytochrome c-conducting Pores in Pure Lipid Membranes

Abstract

During apoptotic cell death, cells usually release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. If Bcl-2 family proteins induce such release by increasing outer mitochondrial membrane permeability, then the pro-apoptotic, but not anti-apoptotic activity of these proteins should correlate with their permeabilization of membranes to cytochrome c. Here, we tested this hypothesis using pro-survival fulllength Bcl-x L and pro-death Bcl-x L cleavage products (⌬N61Bcl-x L and ⌬N76Bcl-x L ). Unlike Bcl-x L , ⌬N61Bcl-x L and ⌬N76Bcl-x L caused the release of cytochrome c from mitochondria in vivo and in vitro. Recombinant ⌬N61Bcl-x L and ⌬N76Bcl-x L , as well as Bcl-x L , cleaved in situ by caspase 3-possessed intrinsic pore-forming activity as demonstrated by their ability to efficiently permeabilize pure lipid vesicles. Furthermore, only ⌬N61Bcl-x L and ⌬N76Bcl-x L , but not Bcl-x L , formed pores large enough to release cytochrome c and to destabilize planar lipid bilayer membranes through reduction of pore line tension. Because Bcl-x L and its C-terminal cleavage products bound similarly to lipid membranes and formed oligomers of the same size, neither lipid affinity nor proteinprotein interactions appear to be solely responsible for the increased membrane-perturbing activity elicited by Bcl-x L cleavage. Taken together, these data are consistent with the hypothesis that Bax-like proteins oligomerize to form lipid-containing pores in the outer mitochondrial membrane, thereby releasing intermembrane apoptogenic factors into the cytosol.Proteins of the Bcl-2 family are key regulators of programmed cell death in multicellular organisms. Some members of this family, including Bax, Bak, Bok/Mtd, Bad, Bik/Nbk, Bid, Blk, Bim/Bod, and Hrk promote apoptosis, whereas others, including Bcl-2, Bcl-x L , Bcl-w, Bfl-1/A1, Mcl-1, and Boo/Diva inhibit apoptosis (1). All these proteins share one to four conserved Bcl-2 homology domains (BH) 1 designated BH1, BH2, BH3, and BH4 (1, 2). In addition, Bcl-2 family members can possess a C-terminal hydrophobic amino acid sequence that helps localize them to intracellular membranes, primarily the outer mitochondrial membrane (1, 2). The activity of Bcl-2 family proteins can be modulated not only at the transcriptional level but also by post-translational modifications (1, 3). For example, various cellular proteases have been shown to cleave Bcl-2, Bcl-x L , Bid, Bax, and Bad producing C-terminal fragments with potent pro-apoptotic activity (4 -23). Bcl-x L can be cleaved by caspase 3 after aspartate 61 and 76 and by calpain after alanine 60, converting Bcl-x L from an antiapoptotic factor to a pro-apoptotic factor (5, 6, 11). Cumulative evidence indicates that Bcl-2 relatives function, at least in part, by regulating the release of proteins enclosed in the mitochondrial intermembrane space. Current models propose that Bcl-2 family proteins exert this function either by forming pores in mitochondrial membranes themselves, or by modulating endogenous mi...

Mitochondrial factors with dual roles in death and survival

“…Although these drugs are generally thought to inactivate anti-death Bcl-2 family proteins, it remains possible that they also activate latent pro-death functions of Bcl-2 and Bcl-xL. In this scenario, the drugs would have an effect similar to proteolytic cleavage of Bcl-2 and Bcl-xL, whereby Bcl-2 and Bcl-xL gain the ability to potently kill cells and to release cytochrome c from intracellular and isolated mitochondria and from pure lipid vesicles (Cheng et al, 1997;Clem et al, 1998;Basan˜ez et al, 2001). An important outcome of the work on various BH3-only proteins (including Bid, Bad, Hrk, Bim, Puma, Noxa and several others) is that we are now armed with the understanding that the binding capacities of Bcl-2 family proteins differ in healthy and dying cells (Suzuki et al, 2000;Wilson-Annan et al, 2003;Jeong et al, 2004).…”

Mitochondrial fission proteins regulate programmed cell death in yeast

“…5C). Like Bcl-xL lacking its C-terminal hydrophobic domain (Basañez et al 2001), ⌬CFis1 became active only when the pH was lowered from pH 7 to pH 5 (Fig. 5D, bottom).…”

“…Nevertheless, we investigated the possibility that yeast Fis1 may share some biophysical properties of Bcl-2/Bcl-xL. Bcl-xL forms pores in synthetic lipid membranes and induces channels on intracellular mitochondria that correlate with its effects on synaptic activity (Basañez et al 2001;Vander Heiden et al 2001;Jonas et al 2003Jonas et al , 2004. Similar to human Bcl-xL, we found that recombinant yeast Fis1 protein efficiently released >80% of 0.4 kDa and 10% of 4 kDa but not larger molecules in a dose-dependent manner from pure lipid vesicles (large unilammelar vesicles, LUV) (Fig.…”

“…LUVs composed of DOPC/DOPG (6/4) at a lipid concentration of 25 µM were prepared in 100 mM KCl, 10 mM Hepes (pH 7.0), and 0.2 mM EDTA, and release of LUV-entrapped fluorescence markers was previously described (Basañez et al 2001). To measure total amount of protein bound to vesicles, LUVs were prepared with D 2 0 instead of H 2 0, facilitating separation of LUV from protein by ultracentrifugation (2 h, 100,000g).…”

Viral versus cellular BCL-2 proteins

“…2,3,46 ± 48 The resulting C-terminal cleavage product is a potent BAX-like pro-apoptotic factor. 3,49,50 Mutation of the caspase cleavage sites causes BCL-2 and BCL-x L to become more potently protective than wild type proteins, arguing that cleavage is an important intracellular event for regulating cell survival/ death. 2,3 Cleavage of BCL-2 may be important in chemotherapy-induced death of leukemia/lymphoma cells, 51,52 and in the death of hepatocytes in mice treated with an antibody to activate the Fas death receptor pathway.…”